INNO-LiPA MYCOBACTERIA (Innogenetics, Ghent, Belgium) is a reverse hybridization DNA probe assay that has been recently improved by increasing the number of identifiable mycobacterial species to 16. Our assessment, performed with 197 mycobacteria belonging to 81 taxa, revealed 100% specificity and sensitivity for 20 out of 23 probes. The probes specific for Mycobacterium fortuitum complex, for the Mycobacterium avium-intracellulare-scrofulaceum group, and for Mycobacterium intracellulare type 2 cross-reacted with several mycobacteria rarely isolated from clinical specimens. The overall sensitivity was 100%, and the overall specificity was 94.4%.